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Gene expression (GE) assays have shown great potential for rapid individual radiation dose exposure assessment. The aim of the present study was to optimise GE-based biological dosimetry protocols for radiological emergencies. Experiments were carried out to validate a newly developed protocol (P2) where several steps were optimised and to compare it with the current validated protocol in place in our laboratory (P1). Several donor blood samples from were exposed ex vivo to of the following doses: 0, 0.5, 1, 2 Gy X-rays. Concomitant measurement of transcription level of genes FDXR, P21, PHPT1, CCNG1 and SESN1 plus HPRT (control) was performed. To summarise, both protocols provided similar dose estimates, P1 being completed in 7 hours while P2 in merely 4 hours. Thus, a significant time shortening was achieved leading to a potential increase of throughput capacity. Hence, this new protocol can be recommended for mass radiation casualties triage purposes., (© The Author(s) 2019. Published by Oxford University Press.)

For triage purposes following a nuclear accident, blood-based gene expression biomarkers can provide rapid dose estimates for a large number of individuals. Ionizing-radiation-responsive genes are regulated through the DNA damage-response pathway, which includes activation of multiple transcription factors. Modulators of this pathway could potentially affect the response of these biomarkers and consequently compromise accurate dose estimation calculations. In the present study, four potential confounding factors were selected: cancer condition, sex, simulated bacterial infection (lipopolysaccharide), and curcumin, an anti-inflammatory/antioxidant agent. Their potential influence on the transcriptional response to radiation of the genes CCNG1 and PHPT1, two biomarkers of radiation exposure ex vivo, was assessed. First, both CCNG1 and PHPT1 were detected in vivo in blood samples from radiotherapy patients and as such were validated as biomarkers of exposure. Importantly, their basal expression level was slightly but significantly affected in vivo by patients' cancer condition. Moreover, lipopolysaccharide stimulation of blood irradiated ex vivo led to a significant modification of CCNG1 and PHPT1 transcriptional response in a dose- and time-dependent manner with opposite regulatory effects. Curcumin also affected CCNG1 and PHPT1 transcriptional response counteracting some of the radiation induction. No differences were observed based on sex. Dose estimations calculated using linear regression were affected by lipopolysaccharide and curcumin. In conclusion, several confounding factors tested in this study can indeed modulate the transcriptional response of CCNG1 and PHPT1 and consequently can affect radiation exposure dose estimations but not to a level which should prevent the biomarkers' use for triage purposes.

Previous investigations in gene expression changes in blood after radiation exposure have highlighted its potential to provide biomarkers of exposure. Here, FDXR transcriptional changes in blood were investigated in humans undergoing a range of external radiation exposure procedures covering several orders of magnitude (cardiac fluoroscopy, diagnostic computed tomography (CT)) and treatments (total body and local radiotherapy). Moreover, a method was developed to assess the dose to the blood using physical exposure parameters. FDXR expression was significantly up-regulated 24 hr after radiotherapy in most patients and continuously during the fractionated treatment. Significance was reached even after diagnostic CT 2 hours post-exposure. We further showed that no significant differences in expression were found between ex vivo and in vivo samples from the same patients. Moreover, potential confounding factors such as gender, infection status and anti-oxidants only affect moderately FDXR transcription. Finally, we provided a first in vivo dose-response showing dose-dependency even for very low doses or partial body exposure showing good correlation between physically and biologically assessed doses. In conclusion, we report the remarkable responsiveness of FDXR to ionising radiation at the transcriptional level which, when measured in the right time window, provides accurate in vivo dose estimates.

Cancer and ionizing radiation exposure are associated with inflammation. To identify a set of radiation-specific signatures of inflammation-associated genes in the blood of partially exposed radiotherapy patients, differential expression of 249 inflammatory genes was analyzed in blood samples from cancer patients and healthy individuals. The gene expression analysis on a cohort of 63 cancer patients (endometrial, head and neck, and prostate cancer) before and during radiotherapy (24 h, 48 h, ~1 week, ~4–8 weeks, and 1 month after the last fraction) identified 31 genes and 15 up- and 16 down-regulated genes. Transcription variability under normal conditions was determined using blood drawn on three separate occasions from four healthy donors. No difference in inflammatory expression between healthy donors and cancer patients could be detected prior to radiotherapy. Remarkably, repeated sampling of healthy donors revealed an individual endogenous inflammatory signature. Next, the potential confounding effect of concomitant inflammation was studied in the blood of seven healthy donors taken before and 24 h after a flu vaccine or ex vivo LPS (lipopolysaccharide) treatment; flu vaccination was not detected at the transcriptional level and LPS did not have any effect on the radiation-induced signature identified. Finally, we identified a radiation-specific signature of 31 genes in the blood of radiotherapy patients that were common for all cancers, regardless of the immune status of patients. Confirmation via MQRT-PCR was obtained for BCL6, MYD88, MYC, IL7, CCR4 and CCR7. This study offers the foundation for future research on biomarkers of radiation exposure, radiation sensitivity, and radiation toxicity for personalized radiotherapy treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, the transcriptional response of a panel of radiation responsive genes was monitored over time in blood samples after radiation exposure in vivo. For this aim, cancer patients treated by radiotherapy were recruited after consent forms were obtained. Following the first fraction of radiotherapy, 2 mL blood samples were collected at different time points during the first 24h hours (before the second fraction was delivered) and at mid and end of treatment. Amongst the 9 genes studied, the gene FDXR stood out as the most sensitive and responsive to the low dose of radiation received from the localised radiation treatment by the circulating white blood cells. The activation of FDXR was found to depend on the volume of the body exposed with a peak of expression around 8–9 hours after irradiation was delivered. Finally results obtained ex vivo confirmed the results obtained in vivo. External beam radiation therapy leads to cellular activation of the DNA damage response (DDR). DNA double-strand breaks (DSBs) activate the ATM/CHEK2/p53 pathway, inducing the transcription of stress genes. The dynamic nature of this transcriptional response has not been directly observed in vivo in humans. In this study we monitored the messenger RNA transcript abundances of nine DNA damage-responsive genes (CDKN1A, GADD45, CCNG1, FDXR, DDB2, MDM2, PHPT1, SESN1, and PUMA), eight of them regulated by p53 in circulating blood leukocytes at different time points (2, 6–8, 16–18, and 24 h) in cancer patients (lung, neck, brain, and pelvis) undergoing radiotherapy. We discovered that, although the calculated mean physical dose to the blood was very low (0.038–0.169 Gy), an upregulation of Ferredoxin reductase (FDXR) gene transcription was detectable 2 h after exposure and was dose dependent from the lowest irradiated percentage of the body (3.5% whole brain) to the highest, (up to 19.4%, pelvic zone) reaching a peak at 6–8 h. The radiation response of the other genes was not strong enough after such low doses to provide meaningful information. Following multiple fractions, the expression level increased further and was still significantly up-regulated by the end of the treatment. Moreover, we compared FDXR transcriptional responses to ionizing radiation (IR) in vivo with healthy donors' blood cells exposed ex vivo and found a good correlation in the kinetics of expression from the 8-hours time-point onward, suggesting that a molecular transcriptional regulation mechanism yet to be identified is involved. To conclude, we provided the first in vivo human report of IR-induced gene transcription temporal response of a panel of p53-dependant genes. FDXR was demonstrated to be the most responsive gene, able to reliably inform on the low doses following partial body irradiation of the patients, and providing an expression pattern corresponding to the % of body exposed. An extended study would provide individual biological dosimetry information and may reveal inter-individual variability to predict radiotherapy-associated adverse health outcomes. [ABSTRACT FROM AUTHOR]

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers. [ABSTRACT FROM AUTHOR]

Though radiotherapy is a local therapy, it has systemic effects mainly influencing immune and inflammation processes. This has important consequences in the long-term prognosis and therapy individualization. Our objective was to investigate immune and inflammation-related changes in the peripheral blood of head and neck cancer patients treated with radiotherapy. Peripheral blood cells, plasma and blood cell-derived RNA were isolated from 23 patients before and at two time points after radiotherapy and cellular immune parameters, plasma protein changes and gene expression alterations were studied. Increased regulatory T cells and increased CTLA4 and PD-1 expression on CD4 cells indicated an immune suppression induced by the malignant condition, which was accentuated by radiotherapy. Circulating dendritic cells were strongly elevated before treatment and were not affected by radiotherapy. Decreased endoglin levels in the plasma of patients before treatment were further decreased by radiotherapy. Expression of the FXDR, SESN1, GADD45, DDB2 and MDM2 radiation-response genes were altered in the peripheral blood cells of patients after radiotherapy. All changes were long-lasting, detectable one month after radiotherapy. In conclusion we demonstrated radiotherapy-induced changes in systemic immune parameters of head and neck cancer patients and proposed markers suitable for patient stratification worth investigating in larger patient cohorts. [ABSTRACT FROM AUTHOR]